Preparation of TGF-beta1/affinity-bound alginate macroporous scaffolds

Tali Tavor Re'em


Spatio-temporal presentation of growth factors is one of the key attributes of the cell's microenvironment. The design of macroporous alginate scaffolds, wherein TGF-b1 or BMP-4 is electrostatically bound to affinity binding sites of alginate sulfate, mimicking their presentation by the extracellular matrix (ECM), was previously shown to enable sustained presentation and release of each factor, thus increasing their biological activity. Specifically, TGF-b1/affinity-bound scaffolds induced the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) seeded within these scaffolds. The prolonged activity of the affinity-bound TGF-b1 enabled efficient induction of signaling pathways leading to chondrogenesis, up to the appearance of committed chondrocytes. Similarly, BMP-4 affinity-bound to the macroporous alginate scaffold enabled efficient induction of osteogenic differentiation in hMSC constructs. Subsequent construction of a multicompartment inductive system, spatially-presenting TGF-b1 and BMP-4 in two distinct layers, enabled complete differentiation of hMSC to chondrocytes and osteoblasts, depending on the type of factor in use in the respective layer.

This paper describes in detail the preparation method of the TGF-b1 or BMP4/ affinity-bound alginate scaffolds, and the set of analyses performed to characterize the resultant scaffolds, including release profile study, released factor bioactivity, and functionality of the scaffolds as hMSC-inductive scaffolds.



alginate; alginate-sulfate; macroporous scaffold; TGF-1; affinity binding; protocol

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Mann BK, Schmedlen RH, West JL. Tethered-TGF-beta increases extracellular matrix production of vascular smooth muscle cells. Biomaterials. 2001;22:439-444.

Park H, Temenoff JS, Tabata Y, Caplan AI, Mikos AG. Injectable biodegradable hydrogel composites for rabbit marrow mesenchymal stem cell and growth factor delivery for cartilage tissue engineering. Biomaterials. 2007;28:3217-3227.

Fan H, Hu Y, Qin L, Li X, Wu H, Lv R. Porous gelatin-chondroitin-hyaluronate tri-copolymer scaffold containing microspheres loaded with TGF-beta1 induces differentiation of mesenchymal stem cells in vivo for enhancing cartilage repair. J Biomed Mater Res A. 2006;77:785-794.

Metzger W, Grenner N, Motsch SE, Strehlow R, Pohlemann T, Oberringer M. Induction of myofibroblastic differentiation in vitro by covalently immobilized transforming growth factor-beta(1). Tissue Eng. 2007;13:2751-2760.

Kopesky PW, Vanderploeg EJ, Kisiday JD, Frisbie DD, Sandy JD, Grodzinsky AJ. Controlled delivery of transforming growth factor beta1 by self-assembling peptide hydrogels induces chondrogenesis of bone marrow stromal cells and modulates Smad2/3 signaling. Tissue Eng Part A. 2011;17:83-92.

Bratt-Leal AM, Carpenedo RL, Ungrin MD, Zandstra PW, McDevitt TC. Incorporation of biomaterials in multicellular aggregates modulates pluripotent stem cell differentiation. Biomaterials. 2011;32:48-56.

Ravindran S, Roam JL, Nguyen PK, Hering TM, Elbert DL, McAlinden A. Changes of chondrocyte expression profiles in human MSC aggregates in the presence of PEG microspheres and TGF-beta3. Biomaterials. 2011; 32: 8436–8445.

Hodneland CD, Lee YS, Min DH, Mrksich M. Selective immobilization of proteins to self-assembled monolayers presenting active site-directed capture ligands. Proc Natl Acad Sci U S A. 2002;99:5048-5052.

Merrett K, Griffith CM, Deslandes Y, Pleizier G, Dube MA, Sheardown H. Interactions of corneal cells with transforming growth factor beta 2-modified poly dimethyl siloxane surfaces. J Biomed Mater Res A. 2003;67:981-993.

Bishop JR, Schuksz M, Esko JD. Heparan sulphate proteoglycans fine-tune mammalian physiology. Nature. 2007;446:1030-1037.

Chen BL, Arakawa T, Hsu E, Narhi LO, Tressel TJ, Chien SL. Strategies to suppress aggregation of recombinant keratinocyte growth factor during liquid formulation development. J Pharm Sci. 1994;83:1657-1661.

Shriver Z, Liu D, Sasisekharan R. Emerging views of heparan sulfate glycosaminoglycan structure/activity relationships modulating dynamic biological functions. Trends Cardiovasc Med. 2002;12:71-77.

Casu B, Lindahl U. Structure and biological interactions of heparin and heparan sulfate. Adv Carbohydr Chem Biochem. 2001;57:159-206.

Raman R, Sasisekharan V, Sasisekharan R. Structural insights into biological roles of protein-glycosaminoglycan interactions. Chem Biol. 2005;12:267-277.

Forsten-Williams K, Chua CC, Nugent MA. The kinetics of FGF-2 binding to heparan sulfate proteoglycans and MAP kinase signaling. J Theor Biol. 2005;233:483-499.

Park JS, Woo DG, Yang HN, Lim HJ, Chung HM, Park KH. Heparin-bound transforming growth factor-beta3 enhances neocartilage formation by rabbit mesenchymal stem cells. Transplantation. 2008;85:589-596.

Park SH, Choi BH, Park SR, Min BH. Chondrogenesis of rabbit mesenchymal stem cells in fibrin/hyaluronan composite scaffold in vitro. Tissue Eng Part A. 2011;17:1277-1286.

Ahmed TA, Giulivi A, Griffith M, Hincke M. Fibrin glues in combination with mesenchymal stem cells to develop a tissue-engineered cartilage substitute. Tissue Eng Part A. 2011;17:323-335.

Benoit DS, Anseth KS. Heparin functionalized PEG gels that modulate protein adsorption for hMSC adhesion and differentiation. Acta Biomater. 2005;1:461-470.

Benoit DS, Collins SD, Anseth KS. Multifunctional hydrogels that promote osteogenic hMSC differentiation through stimulation and sequestering of BMP2. Adv Funct Mater. 2007;17:2085-2093.

Oschatz C, Maas C, Lecher B, Jansen T, Bjorkqvist J, Tradler T, et al. Mast cells increase vascular permeability by heparin-initiated bradykinin formation in vivo. Immunity. 2011;34:258-268.

Freeman I, Kedem A, Cohen S. The effect of sulfation of alginate hydrogels on the specific binding and controlled release of heparin-binding proteins. Biomaterials. 2008;29:3260-3268.

Freeman I, Cohen S. The influence of the sequential delivery of angiogenic factors from affinity-binding alginate scaffolds on vascularization. Biomaterials. 2009;30:2122-2131.

Ruvinov E, Leor J, Cohen S. The effects of controlled HGF delivery from an affinity-binding alginate biomaterial on angiogenesis and blood perfusion in a hindlimb ischemia model. Biomaterials. 2010;31:4573-4582.

Ruvinov E, Leor J, Cohen S. The promotion of myocardial repair by the sequential delivery of IGF-1 and HGF from an injectable alginate biomaterial in a model of acute myocardial infarction. Biomaterials. 2011;32:565-578.

Dvir T, Kedem A, Ruvinov E, Levy O, Freeman I, Landa N, et al. Prevascularization of cardiac patch on the omentum improves its therapeutic outcome. Proc Natl Acad Sci U S A. 2009;106:14990-5.

Re'em T, Kaminer-Israeli Y, Ruvinov E, Cohen S. Chondrogenesis of hMSC in affinity-bound TGF-beta scaffolds. Biomaterials. 2012;33:751-761.

Re'em T, Witte F, Willbold E, Ruvinov E, Cohen S. Simultaneous regeneration of articular cartilage and subchondral bone induced by spatially presented TGF-beta and BMP-4 in a bilayer affinity binding system. Acta biomaterialia. 2012;8:3283-3293.

Amitay-Shaprut S, Freeman I, Cohen S. Affinity-Binding Alginate Scaffolds for the Controlled Delivery of Multiple Heparin-Binding Proteins. In: Berthiaume F, Morgan J, editors. Methods in Bioengineering: 3D Tissue Engineering Methods in Bioengineering. Norwood MA: Artech House; 2010: 101-119.

Kohan M, Breuer R, Berkman N. Osteopontin induces airway remodeling and lung fibroblast activation in a murine model of asthma. Am J Respir Cell Mol Biol. 2009;41:290-296.

Xiao H, Ma X, Feng W, Fu Y, Lu Z, Xu M, et al. Metformin attenuates cardiac fibrosis by inhibiting the TGFbeta1-Smad3 signalling pathway. Cardiovasc Res. 2010;87:504-513.

Re'em T, Tsur-Gang O, Cohen S. The effect of immobilized RGD peptide in macroporous alginate scaffolds on TGFbeta1-induced chondrogenesis of human mesenchymal stem cells. Biomaterials. 2010;31:6746-6755.

Bills CE, Eisenberg H, Pallante SL. Complexes of organic acids with calcium phosphate: the von Kossa stain as a clue to the composition of bone mineral. Johns Hopkins Med J. 1971;128:194-207.


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